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0032-0889/80/65/0880/04/$00.50/0. Light and the Correlation of Chloroplast Development and. Coupling of Phosphorylation to Electron Transport'. Received for ...
Plant Physiol. (1980) 65, 880-883 0032-0889/80/65/0880/04/$00.50/0

Light and the Correlation of Chloroplast Development and Coupling of Phosphorylation to Electron Transport' Received for publication September 27, 1979 and in revised form December 3, 1979

MURRAY E. DUYSEN, THOMAS P. FREEMAN, AND RONALD D. ZABROCKI Department of Botany, North Dakota State University, Fargo, North Dakota 58105 MATERIALS AND METHODS Seedling Growth. Wheat (Triticum aestivum L. cv. 'Waldron') Coupling of phosphorylation to electron transport was examined by measuring the photosynthetic control ratio for broken wheat plastids seeds were germinated in dark at 25 C and high RH (=80%o) in isolated from seedlings at different greening stages. The photosynthetic Vermiculite wetted with 0.5 Hoagland nutrient solution. After 7 control ratio progressively increased during greening and tight coupling days, the etiolated seedlings were exposed to either continuous was noted after granal stacking and thylakoid elongation. ADP impaired light of 350 ,tE m-2s-' (cool-white fluorescent, type F72T12, nonphosphorylating (state 2) electron transport rates of plastids at ex- supplemented with 40 w incandescent) or to intermittent light (2tremely early stages of greening and interfered with photosynthetic control 5 ,uE m-2s-'; 40 w incandescent) for 2 min every 2 h for 2 days measurements. Partially developed plastids exhibited low nonphosphory- following the method of Armond et al. (2). In the continuous light experiments, samples were taken following light exposures of 6, 9, lating electron flow rates but did not exhibit high phosphorylating or 12, 18, 24, 48, and 72 h. Photon flux density was measured with uncoupled electron transport rates to the same extent as nearly developed a Lamda Instruments LI-185 meter. plastids. Prolamellar body dispersal, primary thylakoid production, and the Electron Transport Rates. A 15-g sample of primary leaves was development of photosynthetic control were stimulated equally by 48 homogenized in a Sorvall Omnimixer at half-line voltage for 10 minutes of low irradiance, in cycles of 2 minutes every 2 hours, or by 9 s in 80 ml of cold buffer (pH 8.4) containing: 0.3 M sorbitol, 10 hours of continuous light of moderate irradiance. Wheat plastids that mM KCI, 50 mm Tricine, and 10 mg/ml BSA. The macerate was greened for 6 hours in continuous light of moderate intensity did not filtered through Miracloth and centrifuged in the cold at 270g for exhibit photosynthetic control or much differentiation beyond the etioplast 2 min. The supernatant solution was centrifuged for 5 min at stage. It is concluded that plastid differentiation and the development of 3,000g and the pellet was suspended in cold buffer (pH 8.4) photosynthetic control early in greening under continuous light were limited containing: 0.3 M sorbitol, 10 mm KCI, and 50 mm Tricine. Plastid by developmental time (dark time) rather than by either light intensity or electron transport rates were calculated from the 02 consumed using a Clark-type 02 electrode (Yellow Springs Instruments duration. model 53). Temperature of the reaction medium was maintained at 25 C by circulating water from a constant temperature bath through a glass vessel jacketing the 5-ml reaction vessel. Chloroplasts were irradiated with light from two 300-w projector lamps that passed through two pairs of Coming CS 1-75 and CS 2-58 filters. Light at the center of the reaction vessel was 500 w/m2 as Coupling of phosphorylation to electron transport during plas- measured with a YSI Model 65A radiometer. The reaction metid development has been studied by measuring P/e22 ratios at dium (pH 8.4) contained: 0.3 M sorbitol, 2.5 mM MgCl2, 2.5 mM various stages of development (9, 13). Barley plastids were poorly KCI, 0.1 mm methylviologen, 10 mm KH2PO4, 0.5 mM NaN3, 50 coupled after 48-h greening with NADP as the electron acceptor mM Tricine, and plastids (with a concentration of 10-60 ,ug Chl/ (13) but bean plastids were coupled after 15 h light with ferricya- ml). The reaction mixture was prepared under dim light and nide as the Hill oxidant (9). Coupling of phosphorylation to allowed to temperature equilibrate. Plastids in the vessel were noncyclic electron transport can be determined from photosyn- irradiated and the electron transport rate (state 2) was monitored thetic control ratios (14, 16). Photosynthetic control is calculated for about 20 s before the addition of 0.2 mm ADP (state 3 electron rate). In some experiments, ADP was added to the from the ratio of the electron transport rate during phosphoryla- transport reaction medium dim light, the plastids were irradiated for the tion of ADP (state 3) to the electron transport rate after complete state 3 assay, and instate 4 electron flow was monitored after ADP utilization of ADP (state 4). Here we report the coupling of was depleted. A second ADP aliquot resulted in a repeat state 3 phosphorylation to electron transport in wheat plastids during electron transport rate that was uncoupled by 0.5 mm methylagreening as indicated by photosynthetic control ratios and corre- mine. Chl was determined according to Arnon's technique (3) and late the tightness of coupling with chloroplast development. Fur- electron transport rates were expressed on a per mg Chl basis. thermore, the role of light in determining the photosynthetic Photosynthetic control was calculated from the ratio of the state control ratio was examined by comparing plastid activity of wheat 3 electron transport rate to the state 4 electron transport rate. greened under low intensity intermittent light to plastid activity of Electron transport stimulation was the per cent increase of the wheat greened under continuous light of moderate intensity. state 3 electron transport rate over the state 2 rate (10). Mean electron flow rates and SE were calculated from data of six ' A contribution of North Dakota Agricultural Experiment Station. experiments and plastid preparation measurements were replicated for each experiment. Published with approval of the Director as Journal Paper No. 1020. 2 Abbreviation: P/e2: ratio of P incorporated into ATP per electron pair Electron Microscopy. Sections were taken 1 cm from the tip of primary leaves and were prepared for transmission electron mitransferred. ABSTRACT

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techniques of Freeman and Duysen (6). Chloroplasts of palisade mesophyll cells were examined near the middle vascular bundle using an AEI Corinth electron microscope. croscopy using

RESULTS

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sively decrease electron transport rates over greening (5, 8). The electron transport rates for partially developed wheat plastids were high when expressed on a per mg Chl basis relative to the electron transport rates for nearly developed plastids (Table I). The rates of electron transport for wheat chloroplasts over the first 2 days of greening relative to the electron transport rate for 72 h developed chloroplasts are given in Table I. The reciprocal of Chl values for wheat plastids at different stages of development relative to Chl for 72 h developed plastids are also shown. Comparisons of relative electron transport rates per unit Chl to the reciprocal of relative Chl values were made to note whether decreasing electron transport activity over greening correlated with Chl accumulation. The relative nonphosphorylating electron transport values (state 2 and state 4) were similar over greening to the reciprocal relative Chl values. Early in greening, the relative values for uncoupled and phosphorylating (state 3) electron flow were low compared to reciprocal Chl values. Relative electron transport values for plastids that greened for 24 h or longer were similar to reciprocal Chl values. Plastid Structure. Figure 2 is an electron micrograph of a wheat plastid that developed for 6 h in a seedling leaf under continuous light. The majority of plastids had loose prolamellar bodies, vesiculated primary thylakoids, and a few double thylakoid re-

Electron Transport. Photosynthetic control was observed for wheat chloroplasts isolated from seedlings exposed to continuous light for 9 h or longer (Table I). Consistent with data from studies using pea or spinach plastids (14, 16), the electron transport rate of wheat chloroplasts increased in response to ADP (state 3) but slowed after complete phosphorylation (state 4) and increased again after a second ADP addition (repeat state 3). ADP stimulated an increase in the state 2 rate (electron transport in absence of ADP or basal electron transport). Coupling of phosphorylation to electron transport was further evidenced for wheat plastids by the increased rates of electron flow in the presence of methylamine. The photosynthetic control ratios and the electron transport stimulation percentages progressively increased during early stages of greening and high values were observed for chloroplasts after 24-h development (Table I). The electron transport rate of plastids that greened for 6 h decreased after about 1 min exposure to actinic light in the presence of ADP (Fig. 1, upper trace) and the electron flow rate was neither stimulated again by subsequent gions. Photosynthetic control values and the amount of grana stacking ADP addition nor maintained by a 10-fold higher ADP concentration. ADP impaired electron transport when added during are presented in Figure 3 for wheat seedlings over greening. physiological state 2 (Fig. 1, middle trace) and methylamine Plastids that developed for 9 h in continuous light possessed uncoupled electron transport of 6-h developed plastids in absence primary thylakoids and numerous grana stacks each having about four short thylakoids. After 24 h continuous light, the thylakoid of ADP (Fig. 1, lower trace). Illumination of plastids under nonphosphorylating conditions number per granum was almost twice the thylakoid number per has been suggested to damage subsequent electron transport under granum after 9 h light. Plastid coupling increased by about 30% phosphorylating conditions (16). Our data of electron transport between 9 and 24 h light as indicated by photosynthetic control for wheat chloroplasts support this view. Physiological state 3 rates values. Wheat chloroplast coupling and thylakoid stacking inand photosynthetic control ratios were high in absence of an initial creased by another 25% between 24 and 48 h greening. state 2 rate relative to state 3 rates and photosynthetic control Argyroudi-Akoyunoglou et al. (1) and Armond et al. (2) have ratios noted when preceded by a state 2 rate (Table I). Repeat shown that plastids that green under short duration intermittent state 3 rates of wheat plastids were about 20%o lower than the light have reduced granal stacking but exhibit coupled electron initial state 3 rates. transport (2). We found that wheat chloroplasts greened by low Electron transport becomes functional early in the etioplast to intensity intermittent light (2 min every 2 h for 2 days) exhibited chloroplast conversion but Chl accumulates over an extended electron transport rates and photosynthetic control ratios (Fig. 4) period to increase the size of photosynthetic units and progres- similar to those observed for plastids that greened for 9 or 12 h Table I. Electron Transport Rates, Electron Transport Stimulation, Photosynthetic Control, and Relative Chl of Wheat Chloroplasts at D!fferent Developmental Stages Physiological state 2 is the electron transport rate in absence of ADP; state 3 is the rate during ADP phosphorylation; state 4 represents the rate after complete phosphorylation of ADP. Electron transport stimulation (ETS) is the percentage of increase of state 3 rate over state 2 rate. Photosynthetic control (PC) is the ratio of state 3 to state 4 rates. Each value represents the mean of at least six experiments with two replicates each. The maximum SE was ±5%. Statea State' Relative Rate Statec ReciproLight ETS PC cal RelaTime

2 3 ,ueq/mg Chl-h

3

4

R3

Unc

2

3

4

Unc tive Chld

% ratio ratio u.eq/mg Chl.h ratio 821 21 894 472 563 931 1.9 2.9 1.7 2.4 1.3 2.7 688 30 753 421 545 1041 1.8 2.2 1.4 2.1 1.4 2.3 559 53 632 277 470 750 2.5 1.5 1.2 1.4 1.0 1.5 562 80 616 248 453 747 2.5 1.3 1.2 1.3 1.0 1.1 519 111 546 177 427 658 3.1 1.0 1.0 0.9 0.9 1.0 505 113 528 197 444 718 2.7 1.0 1.0 1.0 1.0 1.0 aPhysiological state 2 was measured prior to state 3 on the same sample. b Measurements were obtained sequentially on the same sample beginning with state 3. Unc is the uncoupled electron transport rate. R3 is the repeat state 3 rate. c Electron transport rates for wheat plastids at different stages of development relative to the electron transport rate of 72-h greened plastids. d Reciprocal of Chl values for wheat plastids at different stages of development relative to Chl for 72-h greened plastids. Chl values were determined by measuring mg Chl/g fresh weight leaf tissue. h 9 12 18 24 48 72

680 527 365 312 246 236

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DUYSEN, FREEMAN AND ZABROCKI

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Physiol. Vol. 65, 1980

*8

Mz 7 z

=6 TIME

n

O5

FIG. 1. Noncyclic electron transport (umol 02/mg Chl-h) of wheat plastids for 6-h greened seedlings. Top trace: ADP (1 ,umol) was added to plastids in a 5-ml reaction vessel prior to light (solid arrow) and in light (first open arrow). Methylamine (25 Jlmol) was added (second open arrow) to uncouple electron transport. Middle trace: ADP (I ,umol) and methylamine (25 Mmol) were added to plastids in light (open arrows). Bottom trace: plastids were uncoupled by methylamine (25 ttmol); ADP was

0 ci a

excluded.

4

0

9

12

48

24

HOURS LIGHT FIG. 3. Histogram summarizing grana stacking (thylakoids/granum) and coupling of phosphorylation to electron transport (photosynthetic control ratio, PC) of wheat plastids over greening. Small bar represents SE.

4 383

F -..

bo

.

e :,

:' ,.

it-

T. -,: ,

.

PC: 1.88 .pl

02

207

ADP

FIG. 2. Wheat mesophyll plastid that differentiated for 6 h in tissue under continuous light. (x 37,000). Inset: vesiculated primary thylakoid of 6-h developed plastid. (x 60,000).

under moderate irradiation (Table I). Low intensity intermittent light induced the conversion of prolamellar bodies to primary thylakoids and stimulated the production of grana each with 2 or 3 short thylakoids in wheat chloroplasts (Fig. 5). This development is somewhat similar to plastids greened for 9 h in continuous light (6).

75 n moles

MA

1 min

355\ TIME

DISCUSSION

FIG. 4. Noncyclic electron transport (Lmol 02/mg Chl-h) and photosynthetic control of wheat plastids that greened under cycles of intermittent light (2 min every 2 h for 2 days).

Coupling of phosphorylation to electron transport progressively increased in wheat seedling plastids during the first 24 h greening as indicated by photosynthetic control ratios (Table I). Wheat plastids that developed mostly primary thylakoids were poorly coupled (Table I, and Figs. 1-5) but coupling became tight after granal stacking and thylakoid elongation (Fig. 3). Nielsen et al. (12) reported tight coupling for barley plastids that had primary thylakoids but were deficient in grana. Apparently, coupling of primary thylakoids in wheat was slow and occurred during the granal stacking stage. The failure to detect photosynthetic control early in wheat greening (Fig. 1) could be related to either insufficient plastid membrane development or membrane fragileness. Plastids after 6-h greening were active in transporting electrons from water to methyl viologen (Fig. 1) and likely possessed coupling factor

particles. Assembly of components for electron transport occurred at extremely early stages of barley greening (5, 8) and coupling factor particles were present in Avena etioplasts (15). Numerous vesiculated membranes were extruded from prolamellar bodies (Fig. 2) of etiochloroplasts. The extent of coupling in wheat plastids could be related to the ability to develop the proper transmembrane proton gradient or form ATP. Methylamine increased electron transport slightly in plastids that failed to exhibit photosynthetic control (Fig. 1). Apparently, undeveloped primary thylakoids of wheat plastids developed a transmembrane proton gradient that was sufficient to limit electron transport but insufficient for detection of photosynthetic control. Arntzen et al. (4) reported a greater proton transport across plastid membranes as the chloroplasts developed. The response of etiochloroplasts to ADP (Fig. 1) could result from an altered conformation of cou-

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LIGHT AND COUPLED ELECTRON FLOW

4-r

i.-Id

FIG. 5. Mesophyll chloroplast from seedlings exposed to intermittent light (cycles of 2 min every 2 h for 2 days). (x 27,600). Inset: limited thylakoid stacks in plastics that developed under intermittent light. (x 80,000).

883

mary thylakoids (Fig. 5) that exhibited photosynthetic control (Fig. 4) equal to the photosynthetic control ratios of plastids from seedlings exposed to 9 or 12 h of more intense continuous light (Table I). Compared to wheat that developed under intermittent light, wheat that greened for 6 h by continuous light should have been exposed to sufficient total irradiance for plastid development and coupling. Plastids greened by 6 h light lacked the extended dark developmental time of the intermittent light treatment and did not exhibit either photosynthetic control or much differentiation beyond etioplasts (Figs. I and 2). The activity of plastids that develop after a short duration of continuous light and varying periods of darkness was not determined since the plastids revert to the prolamellar stage. Early in plastid development, light must be repeated at frequent intervals to prevent reversion to the prolamellar stage (7) and low intensity light prevents the reversion processes. About 9 h of continuous light of moderate intensity is required for complete production of primary thylakoids from prolamellar bodies and for the development of plastids with low photosynthetic control ratios. Proteins synthesized on cytoplasmic ribosomes appear necessary at this time for plastid development (7). Acknowledgment-We are grateful to Linda L. Olson for technical assistance.

pling factor upon binding ADP that reduced membrane permeability to protons. ADP stimulated proton uptake by reducing leakage through coupling factor and the increased proton pressure decreased plastid electron transport rates (11). Chloroplasts of this study were broken during isolation as determined by ferricyanide penetration and electron micrographs. The membrane condition of etiochloroplasts was not examined. Studies are in progress to determine if the isolation of intact etiochloroplasts results in tighter coupling of phosphorylation to electron transport than broken etiochloroplasts. The high electron flow rates of short greening times relative to the rates at long greening times (Table I) were probably due to small photosynthetic unit sizes in partially developed plastids. In this study, electron transport rates were determined from 02 evolved per unit Chl. Electron transport rates for partially developed barley plastids were high when expressed on a per mg Chl basis and these rates decreased during greening as Chl accumulated in photosynthetic units (5, 8). Little Chl accumulated in photosynthetic units of wheat plastids at greening times longer than 24 h (Table I). The relative values for nonphosphorylating electron transport rates of partially developed plastids to 72 h developed plastids correlated well with the inverse of the relative Chl values for partially developed plastids to nearly developed plastids (Table I). This close correlation suggested that the nonphosphorylating electron transport rates of wheat plastids decreased as Chl accumulated in photosynthetic units. The relative rates of phosphorylating (state 3) electron flow were low compared to the reciprocal of the relative Chl values early in greening. We conclude that partially developed wheat plastids are capable of low nonphosphorylating electron flow rates but not high phosphorylating or uncoupled electron transport rates to the same extent as nearly developed plastids. The importance of dark time for the development of plastids and coupling was demonstrated by comparing greening of wheat under intermittent light with greening under continuous light. Even though wheat seedlings were exposed to a total of 48 min of extremely low irradiation, in cycles of 2 min every 2 h, the illumination was adequate for development of plastids with pri-

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LITERATURE CITED ARGYROUDI-AKoYUNOGLOU JH, Z FELEKI, G AKOYUNOGLOU 1971 Formation of two chlorophyll-protein complexes during greening of etiolated bean leaves. Biochem Biophys Res Commun 45: 606-614 ARMOND PA, CJ ARNTZEN, JM BRIANTAIS, C VERNOTTE 1976 Differentiation of chloroplast lamellae. Light harvesting efficiency and grana development. Arch Biochem Biophys 175: 54-63 ARNON DI 1949 Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol 24: 1-15 Arntzen CH, RA Dilley, J Newman 1971 Localization of photophosphorylation and proton transport activities in various regions of the chloroplast lamellae. Biochim Biophys Acta 245: 409-424 BOARDMAN NK 1977 Development of chloroplast structure and function. In A Trebst, M Avron, eds, Encyclopedia of Plant Physiology, New Series Vol 5. Springer-Verlag, Berlin, pp 581-600 FREEMAN TP, ME DUYSEN 1975 The effect of imposed water stress on the development and ultrastructure of wheat chloroplasts. Protoplasma 83: 131-

145 7. FREEMAN TP, ME DUYSEN 1978 The significance of protein synthesis early in the etioplast to chloroplast conversion of wheat leaves as demonstrated by cycloheximide treatment. Protoplasma 97: 111-114 8. HENNINGSEN KW, NK BOARDMAN 1973 Development of photochemical activity and the appearance of the high potential form of cytochrome b-559 in greening barley seedlings. Plant Physiol 51: 1117-1126 9. HOWES CD, Al STERN 1973 Photophosphorylation during chloroplast development in red kidney bean. II. Photophosphorylation and photoreduction appear concomitantly but initially are uncoupled. Plant Physiol 51: 386-390 10. KAMINSKI K 1977 Photosynthetic control in chloroplast suspensions frozen in liquid nitrogen in the presence of glycerol. Z Naturforsch 32c: 254-256 11. MCCARTY RE, JS FUHRMAN, Y TAUCHIYA 1971 Effects of adenine nucleotides on hydrogen-ion transport in chloroplasts. Proc Nat Acad Sci USA 68: 25222526 12. NIELSEN NC, RM SMILLIE, KW HENNINGSEN, D VON WETTSTEIN, CS FRENCH 1979 Composition and function of thylakoid membranes from grana-rich and grana-deficient chloroplast mutant of barley. Plant Physiol 63: 174-182 13. PHUNG NHU HUNG S, A HOARAU, A MOYSE 1970 Etude de 1'evolution en chloroplastes des plastes etioles d'orge. II. Photophosphorylation et photoreduction du NADP, formation de ferredoxine, en enclairment continu et par L'action d' eclairs. Z Pflanzenphysiol 62: 245-248 14. REEVES SG, DD HALL, H BALTSHEFFSKY 1971 Photosynthetic control in isolated spinach chloroplasts with endogenous and artificial electron acceptors. Biochem Biophys Res Commun 43: 359-366 15. WELLBURN AR 1977 Distribution of chloroplast coupling factor (CF,) particles on plastid membranes during development. Planta 135: 191-198 16. WEST KR, JT WISKICH 1968 Photosynthetic control by isolated pea chloroplasts. Biochemical J 109: 527-532